Application of a Spacer-nick Gene-targeting Approach to Repair Disease-causing Mutations with Increased Safety

The CRISPR/Cas9 system is a powerful tool for gene repair that holds great potential for gene therapy to cure monogenic diseases. Despite intensive improvement, the safety of this system remains a major clinical concern. In contrast to Cas9 nuclease, Cas9 nickases with a pair of short-distance (38–68 bp) PAM-out single-guide RNAs (sgRNAs) preserve gene repair efficiency while strongly reducing off-target effects. However, this approach still leads to efficient unwanted on-target mutations that may cause tumorigenesis or abnormal hematopoiesis. We establish a precise and safe spacer-nick gene repair approach that combines Cas9D10A nickase with a pair of PAM-out sgRNAs at a distance of 200–350 bp. In combination with adeno-associated virus (AAV) serotype 6 donor templates, this approach leads to efficient gene repair with minimal unintended on- and off-target mutations in human hematopoietic stem and progenitor cells (HSPCs). Here, we provide detailed protocols to use the spacer-nick approach for gene repair and to assess the safety of this system in human HSPCs. The spacer-nick approach enables efficient gene correction for repair of disease-causing mutations with increased safety and suitability for gene therapy. Graphical overview


Graphical overview Background
In the CRISPR/Cas9 system, a single-guide RNA (sgRNA)-directed Cas9 nuclease introduces double-strand breaks (DSBs) at the target region. DSBs are predominantly repaired by the non-homologous end joining (NHEJ) pathway, causing micro-insertions or deletions (indels or unwanted on-target mutations). If a DNA donor template with 5′ and 3′ homology arms (HAs) is provided, the homology-directed repair (HDR) pathway is activated to precisely replace the mutated DNA sequence (Cong et al., 2013;Hsu et al., 2013;Mali et al., 2013b;Chu et al., 2015). Although the CRISPR/Cas9 system succeeded in repairing mutations, its undesired on-target mutations and potential off-target activities are still a major concern (Cradick et al.
CAAGCAGAAGACGGCATACGAGAT tcgcctta GTCTCGTGGGCTCGG   2. Design pools of sgRNAs on the selected areas with a PAM-out configuration using CrispRGold software.

Equipment
In combination with the Cas9 D10A nickase, 5′ sgRNAs nick the plus strand, whereas 3′ sgRNAs nick the opposite strand of the dsDNA molecule. 3. Order designed sgRNAs, clone them into the suitable plasmids, and test gene editing efficiencies of these sgRNAs using T7EI assay or Sanger sequencing following ICE analysis. 4. Select several pairs of PAM-out sgRNAs (one 5′ sgRNA and one 3′ sgRNA) with equal and high gene editing efficiencies (>80%) for the spacer-nick gene repair approach.

B. Design DNA donor templates
Note: In order to quantify gene correction efficiencies, we insert a diagnostic restriction enzyme site by introducing silent mutations into correct sequences or develop correct integration PCR, which allows us to amplify repaired and non-repaired alleles. To clone DNA donor fragments into the AAV genome vector, we inserted NotI sites into both ends of DNA donor fragments together with six random nucleotides. 1. Design a DNA donor template carrying 5′ and 3′ HAs of at least 0.5-1.5 kb outside of each nick site ( Figure  2). 2. In order to minimize a mini-homologous sequence (highlighted in green; Figure 2) between two nick sites, we modify this sequence by introducing silent mutations (in case of repairing mutations in coding exons) or by partially ablating this sequence (in case of inserting cDNA into loci). 3. Add NotI restriction enzyme sites to both ends of the DNA donor template. 4. Order the dsDNA template from IDT.

D. Production and purification of AAV6 donor vectors
Note: We recommend using high-fidelity KOD hot-start DNA polymerase for PCR amplification of the DNA donor template. However, you can use any high-fidelity DNA polymerases that are available in your laboratory. Production and purification of AAV6 donor vectors are described in detail in our previous protocol (Tran et al., 2020). 1. Amplify the NotI-containing dsDNA donor template (step B4) with specific forward and reverse primers (carrying six random nucleotides, NotI site, and target-specific sequence) using any high-fidelity DNA polymerases. 2. Purify PCR products using NucleoSpin Gel and PCR Clean-up kit. 3. Digest PCR products with NotI restriction enzyme and load on 0.8% agarose gel. 4. Cut digested dsDNA band and purify it using any gel extraction kit. 5. Clone the purified dsDNA template into NotI-linearized pAAV vectors using T4 DNA ligase. 6. Transform the cloned pAAV vectors into competent TOP10 bacteria using heat-shock method. 7. Spread transformed bacteria on 10 cm LB agar dishes containing 50 μg/mL of carbenicillin and culture these dishes in a bacterial incubator at 30 °C overnight. 8. Pick bacterial colonies and inoculate these with 2 mL of liquid LB medium containing 50 μg/mL of carbenicillin at 30 °C overnight in a bacterial incubator with shaker. 9. Extract plasmids using any plasmid purification kit and confirm the insert by Sanger sequencing. 10. Produce large quantities of pAAV donor vectors using NucleoBond Xtra Maxi kit.  11. Transfect, purify, concentrate, and calculate copy number of AAV6 donor particles following our previous protocol (Tran et al., 2020).

E. Culture human HSPCs
Note: In order to achieve sufficient expansion of human CD34 + cells, we recommend that you culture ~4 × 10 5 HSPCs per 2 mL in a well of a 6-well plate and monitor the culture every day until these cells are used for electroporation. In case expanded cells are at high density, you should split them into new wells of a 6-well plate.
1. Isolate human CD34 + HSPCs from mobilized peripheral blood or bone marrow of healthy donors or patients using Ficoll-Paque Plus and follow human CD34+ microbead kit according to the manufacturer's protocol. 2. Freeze isolated CD34 + HSPCs in serum-free freezing medium (BAMBANKER) at a density of 1 × 10 6 per milliliter and store in liquid nitrogen for long-term storage. 3. Thaw frozen vials and culture CD34 + HSPCs at a density of 4 × 10 5 per 2 mL of completed serum-free StemSpan TM SFEM II medium in a well of a 6-well plate. 4. Exchange half of the old medium with new medium 24 h post culture. 5. Seventy-two hours post culture, harvest expanded HSPCs and count cell number using TC20 cell counting system. 6. Transfer 2 × 10 5 HSPCs to a 1.5 mL Eppendorf tube and spin down at 300 × g for 5 min. 7. Remove supernatant, wash the cell pellet two times with room temperature PBS, and proceed to Section F or Section G. Anneal two oligos of dsODN by mixing 50 μL of each oligo (100 pmol), incubate at 95 °C for 5 min, and ramp down at a rate of 1 °C/s to room temperature in the thermocycler. 3. After the last wash with PBS in step E7, remove supernatant and resuspend the cell pellet with 20 μL of P3 Primary Cell electroporation buffer by pipetting up and down 10 times. 4. Add 2.75 μL of 5′ and 3′ assembled RNPs (step C5) and 0.5 μL (25 pmol) of the annealed dsODN to cell suspension and mix well by pipetting up and down five times. 5. Transfer the mixture to a well of a 16-well nucleocuvette strip. 6. Electroporate the cells using the DZ-100 program of Lonza 4D-Nucleofector. 7. Transfer the electroporated cells to pre-warmed serum-free StemSpan TM SFEM II medium. 8. Place the cell plate into an incubator at 37 °C and 5% CO2. 9. Change medium every 2-3 days. 10. At day 10 post electroporation, harvest the edited HSPCs by pipetting up and down five times, transfer cell suspension to a 15 mL Falcon tube, and spin down at 300 × g for 5 min. 11. Wash the cell pellet three times with room temperature PBS. 12. Deplete the dead cells by using dead cell removal kit and follow manufacturer's specifications. 13. Isolate gDNA using GenFind V3 reagent kit according to the manufacturer's protocol. 14. Measure concentration of gDNA using NanoDrop and dilute gDNA to 20 ng/μL. 15. Check quality of gDNA by loading 500 ng of gDNA on 0.8% agarose gel ( Figure 3) and proceed to Section H. Note: In order to achieve good results of Tn5-mediated DNA tagmentation, we recommend using GenFind V3 kit for gDNA isolation. This kit yields high quality of gDNA, and DNA fragment size is >20 kb ( Figure  3). However, you can also use other gDNA isolation kits with equal quality that are available in your laboratory.   10. At day 18 post infection, harvest the cells by pipetting up and down five times, transfer cell suspension to a 15 mL Falcon tube, and spin down at 300 × g for 5 min. 11. Wash the cell pellets three times with room temperature PBS. 12. Deplete dead cells by using dead cell removal kit following manufacturer's specifications. 13. Isolate gDNA using Wizard Genomic DNA purification kit (see the note at step F15) and proceed to Section I.

H. Tn5 transposase-mediated GUIDE-seq
Note: Accurate concentration of gDNA is essential for reproducible Tn5-mediated tagmentation. We describe a Tn5-tagmented DNA protocol for exactly 100 ng of gDNA. If you want to conduct the protocol for more or less than 100 ng of gDNA, you should optimize these conditions. The Tn5-mediated DNA tagmentation should produce DNA fragments in the range of 300-1,500 bp. If DNA fragments are smaller or larger than this, you should reduce or increase the incubation time for the tagmentation, respectively. Importantly, you must carry out the Tn5-mediated DNA tagmentation at room temperature; do not use vortex for mixing. Primers used in this protocol are the same as described in Tran et al. (2022). 1. Add the following reagents stepwise to a well of an 8-well PCR strip as in Table 1: 8. Check quality of tagmented DNA fragments by loading 3 µL of the elute on 1.2% agarose gel (see Figure  4) and proceed to step H9. 9. Prepare first PCR in a well of a 96-well plate according to Table 2. h. Repeat steps f-g one more time. i. Let the beads air dry for 3-5 min. j. Take the plate out of the magnetic stand, add 18 μL of nuclease-free water, mix the beads, and incubate at room temperature for 2 min. k. Put the plate on the magnetic stand for 5 min. l. Transfer 15 μL of eluted DNA to a new well of a 96-well PCR plate and proceed to step H12. 12. Prepare second index PCR in a well of a 96-well plate according to Table 3.  16. Verify size of PCR products by loading 3 µL of the eluate on a 1.5% agarose gel ( Figure 5).  28. Extract DNA using QIAquick Gel Extraction kit following the manufacturer's specifications. 29. Elute DNA with 100 μL of nuclease-free water. 30. Pool two lanes into a 2 mL Eppendorf tube. 31. Clean up and concentrate the final DNA library with AMPure XP beads (0.9×). 32. Elute DNA with 30 μL of 1× TE buffer (pH 8.0). 33. Measure DNA concentration of the final DNA library using Qubit dsDNA high sensitivity kit following manufacturer's specifications. 34. Verify size range of the eluted DNA (step H22) and the final DNA library (step H32) using the D1000 ScreenTape kit (Figure 7).

I. LAM-HTGTS
Note: Contamination of low molecular weight DNA fragments (<1 kb) will inhibit the LAM-HTGTS; therefore, it is crucial to remove any <1 kb fragments by cleaning gDNA up with ProNex size-selective purification beads. It is imperative to optimize the linear amplification PCR for it not to amplify any double-stranded products that will block the generation of the linear amplicons and the adapter ligation. 1. Ensure the high quality of gDNA by loading 50-100 ng on 0.8% agarose gel. If any <1 kb fragments are detected (Figure 8), purify the gDNA with 1.0× ProNex size-selective purification beads. 2. Prepare linear amplification PCR using PrimeSTAR GXL DNA polymerase as in Table 4.  a. Transfer 2 μL of Dynabeads TM MyONE TM Streptavidin C1 per each PCR reaction into a 1.5 mL Eppendorf tube and add 150 μL of B&W buffer to the tube. b. Put the tube on the magnetic stand (6-well magnetic rack) for 5 min. c. Remove the supernatant and add 150 μL of B&W buffer to the tube. d. Take the tube out of the magnetic stand and resuspend the beads by pipetting up and down. e. Put the beads on the magnetic stand for 5 min and remove the supernatant. f. Resuspend the beads in 2 μL of nuclease-free water per each PCR reaction and mix the reactions as shown in Table 5. g. Incubate the reaction on the tube roller for 2-4 h. Note: Four hours is recommended, but the reaction can be rolled overnight. h. Capture the DNA-bead complexes on the magnetic stand. i. Remove the supernatant and wash the beads with 150 μL of B&W buffer three times. j. Wash the beads with 150 μL of nuclease-free water. k. Resuspend the beads in 9 μL of nuclease-free water. 6. Proceed with on-bead ligation steps as follows: a. Prepare and mix the reagents as in Table 6. . Load 5 μL of the reaction on 2% agarose gel along with the 100 bp ladder. The peak of the length distribution should be in the range of 300-500 bp (Figure 9).

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Published: Apr 20, 2023 on-bead adapter PCR products. The length distribution of on-bead adapter PCR products (A and B) should be in the range of 300-500 bp.
10. Purify PCR products with ProNex beads (1.2×) and proceed to Illumina library preparation. 11. Tag different samples with Illumina barcode sequences by mixing the reagents as in Table 8:  13. Load 5 μL of the reaction on 2% agarose gel along with the 100 bp ladder. The peak of the length distribution should be around 300-500 bp ( Figure 10). 14. Quantify the concentration of the products 500-700 bp long using densitometry in GelAnalyzer software. 15. Pool the samples so that the mass of the product per mass of gDNA used as an input (or number of the cells) is equal, to ensure a homogenous variant coverage. 16. Load 50-100 μL of the reaction on 0.8% agarose gel along with the 100 bp ladder and cut the 500-1,000 bp smear from the gel. 17. Extract the product from the agarose using QIAquick gel extraction kit. Measure the concentration on Nanodrop.
Published: Apr 20, 2023 18. Ensure the proper size selection by loading the library diluted to 10 ng/μL on High Sensitivity D1000 ScreenTape or High Sensitivity dsDNA chip of BioAnalyzer ( Figure 11). 19. Calculate the molarity of the sample using ScreenTape or BioAnalyzer software and proceed with the library loading as described in the corresponding Illumina kit manufacturer's manual.

Data analysis
For GUIDE-seq experiments, three independent experiments with two replicates each were performed. For LAM-HTGTS experiments, 3-6 independent experiments with two replicates each were performed. The pipelines for analyzing the GUIDE-seq and LAM-HTGTS were based on previous publications (Giannoukos et al., 2018;Danner et al., 2021). Detailed analysis of GUIDE-seq and LAM-HTGTS has been described in the Methods section and the supplementary figure S9 of the original paper (Tran et al., 2022). The Jupiter notebooks, conda environment, and scripts are available on https://github.com/ericdanner/SpacerNick. Briefly, GUIDE-seq reads were checked for correct priming and the sequence of the dsODN was trimmed to adjacent genomic sequences that are globally mapped to human genome (hg38) using Bowtie2. Mapped reads that were aligned to regions within 5,000 bps of the off-predicted target sites were quantified. For LAM-HTGTS analysis, reads were checked for correct priming and then aligned to the AAV ITR sequence to quantify AAV integrations. The unaligned reads were end-to-end mapped to in silico-generated outcomes. Remaining unmapped reads were then trimmed to the Cas9 target sites

50 mM bridge adapter
Anneal two oligonucleotides [sequences are identical to Hu et al. (2016)] by heating the 400 mM (total) mixture in 25 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.5 mM EDTA at 98 °C and ramp down at a rate of 1 °C/min to room temperature using the thermocycler. Dilute the mixture to 50 mM in nuclease-free water, aliquot, and store at -20 °C.